Methods for using monoclonal antibodies specific for cell-surface bound LAM-1

ABSTRACT

A shed form of leukocyte adhesion molecule-1 (LAM-1, L-selectin) is present in high levels in human plasma. Quantitative methods of detecting shed LAM-1 (sLAM-1) by Western blot and ELISA analysis are disclosed. Also disclosed are methods for the specific detection of cell-surface bound LAM-1 in the presence of shed LAM-1 and for immunotherapy using monoclonal antibodies reactive with cell-surface bound LAM-1 but not reactive with shed LAM-1.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/334,191, filedNov. 4, 1994 entitled: SPECIFIC DETECTION OF CELL SURFACE RECEPTORLEUKOCYTE ADHESION MOLECULE-1, which is a divisional of application Ser.No. 07/862,483, filed Apr. 2, 1992 (now U.S. Pat. No. 5,389,520, issuedFeb. 14, 1995) which is a continuation-in-part of Ser. No. 07/730,503,filed Jul. 8, 1991 (now abandoned), which is a Continuation of Ser. No.07/313,109, filed Feb. 21, 1989 (now abandoned) and aContinuation-in-Part of Ser. No. 07/700,773, filed May 15, 1991 (nowabandoned) and a Continuation-in-part of Ser. No. 07/737,092, filed Jul.29, 1991 (now abandoned) and a Continuation-in-Part of Ser. No.07/770,608, filed Oct. 3, 1991, (now abandoned).

FIELD OF THE INVENTION

This invention relates to human leukocyte-associated cell surfaceproteins, particularly to leukocyte adhesion molecule-1 (LAM-1) andmethods for its detection.

Part of the work leading to this invention was made with United StatesGovernment funds. Therefore, the U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

The ability of leukocytes to leave the circulation and to migrate intotissues is a critical feature of the immune response. Normally, theinfiltrating leukocytes phagocytize invading organisms or dead ordamaged cells. However, in pathologic inflammation, infiltratingleukocytes can cause serious and sometimes deadly damage.Leukocyte-mediated inflammation is implicated in a number of humanclinical manifestations, including the adult respiratory distresssyndrome, multi-organ failure and reperfusion injury.

Several different receptor adhesion molecules participate in the processof adhesion and transmigration of leukocytes through vascularendothelium at sites of inflammation (Springer, Nature 346:425-434(1990)). One of the several molecules involved in the initial attachmentof leukocytes to endothelium is the leukocyte adhesion molecule-1(LAM-1, L-selectin) (Kishimoto et al., Proc. Natl. Acad. Sci. USA87:2244-2248 (1990); Ley et al., Blood 77:2553-2555 (1991); Spertini etal., J. Immunol. 147:2565-2573 (1991)). LAM-1 is a member of theselectin family of adhesion molecules (Bowen et al., J. Cell Biol.109:421-427 (1989); Siegelman et al., Proc. Natl. Acad. Sci. USA86:5562-5566 (1989); Tedder et al., J. Exp. Med. 170:123-133 (1989))that includes, the mouse L-selectin, MEL-14 (Gallatin et al., Nature304:30-34 (1983); Lasky et al., Cell 56:1045-1055 (1989); Siegelman etal., Science 243:1165-1172 (1989)), Endothelial-Leukocyte AdhesionMolecule-1 (ELAM-1, E-selectin) (Bevilacqua et al., Proc. Natl. Acad.Sci. USA 84:9238-9243 (1987); Bevilacqua et al., Science 243:1160-1164(1989); Luscinskas et al., J. Immunol. 1422257 (1989); Luscinskas etal., J. Immunol. 146:1617-1625 (1991)), and CD62 (PADGEM, GMP-140,P-selectin) (Geng et al., Nature 343:757-760 (1990); Johnston et al.,Cell 56:1033-1044 (1989); Larsen et al., Cell 59:305-312 (1989); Larsenet al., Cell 63:467-474 (1990)). All selectins are derived fromevolutionarily related genes (Collins et al., J. Biol. Chem.266:2466-2478 (1991); Johnston et al., J. Biol. Chem. 34:21381-21385(1990); Ord et al., J. Biol. Chem. 265:7760-7767 (1990); Watson et al.,J. Exp. Med. 172:263-272 (1990)), and are characterized by an NH₂-terminal, Ca⁺ -dependent lectin domain, an epidermal growth factor(EGF)-like domain followed by multiple short consensus repeat (SCR)domains, a transmembrane region, and a cytoplasmic tail.

LAM-1 is expressed on the surface of most leukocytes, includinglymphocytes, neutrophils, monocytes, eosinophils, hematopoieticprogenitor cells and immature thymocytes (Griffin et al., J. Immunol.145:576-584 (1990); Tedder et al., J. Immunol. 144:532-540 (1990)).LAM-1 is a highly glycosylated protein of 95-105,000 M_(r) onneutrophils and 74,000 M_(r) on lymphocytes (Griffin et al., J. Immunol.145:576-584 (1990); Tedder et al., Eur. J. Immunol. 20:1351-1355(1990)). Human LAM-1 and mouse MEL-14 mediate the binding of lymphocytesto high endothelial venules (HEV) of peripheral lymph nodes throughinteractions with a constitutively expressed ligand (Imai et al., J.Cell Biol. 113:1213-1221 (1991); Kishimoto et al., Proc. Natl. Acad.Sci. USA 87:2244-2248 (1990); Spertini et al., Leukemia 5:300-308(1991); Stamper Jr. et al., J. Exp. Med. 144:828-833 (1976); Tedder etal., J. Immunol. 144:532-540 (1990)), and are also involved inlymphocyte, neutrophil and monocyte attachment at sites of inflammation(Hallmann et al., Biochem. Biophys. Res. Commun. 174:236-243 (1991);Jutila et al., J. Immunol. 143:3318-3324 (1989); Lewinsohn et al., J.Immunol. 138:4313-4321 (1987); Smith et al., J. Clin. Invest. 87:609-618(1991); Spertini et al., J. Immunol. 147:2565-2573 (1991); Watson etal., Nature 349:164-167 (1991)). In vitro, endothelial cell surfaceexpression of the LAM-1 ligand(s) is induced only after exposure of theendothelial cells to inflammatory cytokines, and the endothelial ligandshares many functional features with the LAM-1 ligand(s) expressed byHEV (Smith et al., J. Clin. Invest. 87:609-618 (1991); Spertini et al.,J. Immunol. 147:2565-2573 (1991)). Sulfated carbohydrates and mAb thatbind to the lectin domain of LAM-1 inhibit LAM-1-specific adhesion (Imaiet al., J. Cell Biol. 113:1213-1221 (1991); Kansas et al., J. Cell Biol.114:351-358 (1991); Spertini et al., J. Immunol. 147:2565-2573 (1991);Stoolman et al., Blood 70:1842-1850 (1987); Yednock et al., J. CellBiol. 104:725-731 (1987); Yednock et al., J. Cell. Biol. 104:713-723(1987)). The lectin domain of LAM-1 seems to act as the ligand bindingunit to determine specificity, while the EGF-like and SCR domains appearto regulate the affinity of this interaction (Kansas et al., J. CellBiol. 114:351-358 (1991); Siegelman et al., Cell 61:611-622 (1990);Spertini et al., J. Immunol. 147:942-949 (1991); Watson et al., J. CellBiol. 115:235 (1991)).

It has been proposed that the treatment of a patient suffering frompathologic inflammation with an antagonist to adhesion receptor functioncan result in the reduction of leukocyte migration to a level manageableby the target endothelial cells and the subsequent dramatic recovery ofthe patient. Local administration of therapeutic agents can blockcompetitively the adhesive interactions between leukocytes and theendothelium adjacent to an inflamed region. Therapeutic agents can alsobe administered on a systemic level for the treatment of a patientsuffering from disseminated inflammation (Harlan and Liu, eds.,Adhesion: Its Role in Inflammatory Disease, W. H. Freeman (in press)).

A unique feature of the L-selectins is that both human LAM-1 and mouseMEL-14 are shed from the cell surface following cellular activation invitro (Griffin et al., J. Immunol. 145:576-584 (1990); Jung et al., J.Immunol. 144:3130-3136 (1990); Kishimoto et al., Science 245:1238-1241(1989); Kishimoto et al., Proc. Natl. Acad. Sci. USA 87:2244-2248(1990); Spertini et al., Leukemia 5:300-308 (1991)). It has beenproposed for the mouse that shedding of MEL-14 from leukocytes might benecessary to enable the leukocytes to transmigrate through endotheliuminto sites of inflammation in vivo (Jutila et al., J. Immunol.143:3318-3324 (1989); Kishimoto et al., Science 245:1238-1241 (1989)).This would provide a rapid means for the regulation of leukocyteadhesion and de-adhesion to endothelium. Although the subsequent fateand possible function of the shed LAM-1 (sLAM-1) molecule is not known,the presence of a soluble factor present in rat thoracic duct lymphcapable of inhibiting lymphocyte binding to HEV has been demonstrated(Chin et al., J. Immunol. 125:1764-1769 (1980)). Furthermore, thisfactor was shown to be antigenically related to a structure(s) presenton lymphocytes (Chin et al., J. Immunol. 125:1764-1769 (1980); Chin etal., J. Immunol. 125:1770-1774 (1980); Chin et al., J. Immunol.131:1368-1374 (1983)).

A number of surface molecules present on cells of various lineages arenow known to be shed and thereby released into the extracellular milieu(Tedder, Am. J. Respir. Cell Mol. Biol. 5:305-306 (1991)). These includemany of the growth factor receptors, the receptors for interleukin-1,interleukin-2 (CD25), transferrin (CD72), insulin, growth hormone, tumornecrosis factor (Porteu et al., J. Exp. Med. 172:599-607 (1990)),colony-stimulation factor-1 (Downing et al., Mol. Cell. Biol.9:2890-2896 (1989)) and nerve growth factor (DiStefano et al., Proc.Natl. Acad. Sci. USA 85:270-274 (1988)) as well as CD8, and CD14. Theseproteins are quite diverse in structure and amino acid sequence and haveno unifying functional characteristics that are currently appreciated.In most cases, proteases cleave the receptor near the membrane,releasing a nearly intact extracellular domain (DiStefano et al., Proc.Natl. Acad. Sci. USA 85:270-274 (1988); Downing et al., Mol. Cell. Biol.9:2890-2896 (1989); Kishimoto et al., Science 245:1238-1241 (1989);Spertini et al., Leukemia 5:300-308 (1991)).

No definitive functions for shed receptors have been elucidated,although many of the shed receptors retain ligand-binding activity.Thus, receptor function may be regulated not only by proteolyticcleavage of the receptor from the cell surface, but also by the presenceof shed receptor in the extracellular environment.

If the shed form of LAM-1 retains ligand-binding activity, however, oneconsequence of its presence might be a potential interference withdiagnostic or therapeutic administration of antagonists to LAM-1function. The shed form of the receptor, if present in large amounts,could competitively bind any administered LAM-1 antagonist, thusthwarting the diagnostic effort or the treatment regimen.

SUMMARY OF THE INVENTION

We report here that the shed leukocyte adhesion molecule-1 (sLAM-1) fromboth lymphocytes and neutrophils was demonstrated to be present in highlevels in human plasma. Two assay methods have been used, Western blotanalysis and a specially developed, quantitative enzyme-linkedimmunosorbent assay or ELISA. Semipurified sLAM-1 from plasma inhibitedLAM-1-specific attachment of lymphocytes to cytokine-activatedendothelium in a dose dependent manner. Total inhibition ofLAM-1-dependent lymphocyte attachment was achieved at sLAM-1concentrations of 8 to 15 μg/ml, while physiological concentrations ofsLAM-1 caused a small but consistent inhibition of lymphocyteattachment. sLAM-1 in plasma also inhibited binding of most anti-LAM-1mAb (2 to 5 μg/ml) to the surface of leukocytes. However, one epitopepresent within the EGF-like domain of LAM-1 was lost in sLAM-1,suggesting a conformational change in the structure of the receptorafter shedding.

The inhibition of LAM-1 dependent lymphocyte attachment by the shed formof the receptor, even at physiological concentrations of sLAM-1, and theinhibition of binding of most anti-LAM-1 mAb could present substantialproblems for therapeutic regimens requiring systemic administration ofantagonists to LAM-1 function or for diagnostic techniques. The presenceof sLAM-1 could require that such agents be administered in excessivedoses to be effective. However, our discovery of a difference inepitopes between the EGF-like domains of the LAM-1 molecule and its shedform has permitted the development of diagnostic and therapeutic agentsthat react with cell surface bound receptor LAM-1 without binding to theshed form of the molecule.

In one aspect the invention generally features monoclonal antibodyreactive with LAM-1, and not reactive with shed LAM-1, and a method forisolating the antibody. Preferably, the antibody has the same pattern ofreactivity with LAM-1 as monoclonal antibody anti-LAM1-1; mostpreferably the antibody is anti-LAM1-1 monoclonal antibody. Theinvention also features methods of using the selective antibody indiagnostic and therapeutic applications.

In another aspect, the invention features a method for quantitating theamount of LAM-1 or fragment thereof in a sample that includes reactingthe sample in a binding assay with a binding agent or ligand for LAM-1or fragment thereof, and comparing the results of the binding assay to astandard curve. Preferably, the fragment of LAM-1 to be detected is shedLAM-1; the sample to be assayed is a biological sample, most preferablythe sample is from a patient and the amount of LAM-1 or fragmentdetermined in the binding assay is to be used as a diagnostic variable;the binding assay is either an ELISA or a Western blot; and the bindingagent is an anti-LAM-1 antibody.

In another aspect, the invention features a kit for use in quantitatingthe amount of LAM-1 or fragment thereof in a sample. The kit includescomponents required for extraction of the sample and componentsnecessary for use in a binding assay, including a binding agent orligand for LAM-1 or fragments thereof.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Development of the sLAM-1-ELISA allowed for the first time an easy,efficient and sensitive method for quantification of sLAM-1. The directdemonstration of sLAM-1 in plasma and supernatant fluid from activatedneutrophils and lymphocytes confirms that the in vivo and in vitro lossof LAM-1 leukocyte surface expression is due to shedding. The extent ofshedding is high; in a population of normal healthy blood donors themean serum level of sLAM-1 was determined to be 1.6±0.8 μg/ml.

The shed form of LAM-1 was found to be different from the cellsurface-bound form in one major way. sLAM-1 did not contain the epitopewithin the EGF-like domain of LAM-1 identified by the anti-LAM1-1 mAb,while this epitope is readily demonstrated on cell-surface LAM-1 (Kansaset al., J. Cell Biol. 114:351-358 (1991); Spertini et al., J. Immunol.147:942-949 (1991); Tedder et al., J. Immunol. 144:532-540 (1990)). Thiswas revealed both by ELISA analysis and by a lack of competitiveinhibition in immunofluorescence staining of leukocytes. Since theEGF-like domain is retained in sLAM-1, it is possible that alterationsin the tertiary structure of LAM-1 occur following shedding that resultin a lack of anti-LAM1-1 mAb binding. This loss of reactivity does notappear to be due to technical problems as the LAM-1 epitope was readilydemonstrated to be present on a soluble LAM-IgG fusion protein.

Detection and Characterization of sLAM-1

sLAM-1 in serum appears to be derived from both lymphocytes andneutrophils since the analysis of sLAM-1 isolated from neutrophils(95-105,000 M_(r)) and lymphocytes (74,000 M_(r)) produced bands ofcorresponding M_(r). The differences in M_(r) between lymphocyte andneutrophil sLAM-1 most likely result from differences in glycosylationof a single protein species since only a single LAM-1 mRNA species hasbeen identified (Ord et al., J. Biol. Chem. 265:7760-7767 (1990)).Cleavage of LAM-1 to produce sLAM-1 is likely to be proximal to thetransmembrane region encoded by exon VIII (Ord et al., supra); cleavagein this region would account for the small difference in M_(r) betweenthe intact cellular LAM-1 molecule and its shed form (Kishimoto et al.,Proc. Natl. Acad. Sci. USA 87:2244-2248 (1990); Lasky et al., Cell.56:1045-1055 (1989); Spertini et al., Leukemia 5:300-308 (1991)).

The concentration of sLAM-1 was determined by comparison with areference plasma sample that ranged between 0.85 to 1.9 μg/ml, havingbeen standardized by two independent means. The mean of this range ofvalues of 1.3 μg/ml should represent a conservative estimate of theactual amount of sLAM-1 present in plasma, and was therefore chosen asthe basis of all future calculations. Theoretical considerations basedon experimental data derived from shedding experiments in K562-LAM-1transfectants further substantiate the validity of this estimate.Assuming that 30 to 40,000 LAM-1 molecules are present on each K562-LAMcell, and that all molecules (MW 71,000) are shed following PMAstimulation, the concentration of sLAM in plasma can be calculated bycomparing the concentration of sLAM-1 (1.7% the concentration ofstandard plasma) shed from K562 transfectants (1×10⁷ cells/ml) to atitration curve of standard plasma. According to this calculation,plasma concentrations would lie in the range of 2.1 to 2.8 μg/ml.Similar considerations make it highly unlikely that the amount of sLAM-1found in serum or plasma is generated from the shedding of cell surfaceLAM-1 by blood leukocytes present during the preparation of the samples:the quantity of sLAM-1 actually found in serum is 10 to 25 fold higherthan the quantity of LAM-1 that could maximally be shed from thecell-surface of leukocytes (˜4×10⁶ /ml) present in the samples, assuminga density of 50-100,000 receptors/cell. Therefore, sLAM-1 must begenerated by the ongoing shedding of cell surface LAM-1 from leukocytesin vivo.

sLAM-1 semipurified from plasma inhibited LAM-1-mediated lymphocytebinding to TNF-activated endothelium in a dose dependent manner withcomplete inhibition at ˜8-15 μg/ml. At concentrations of sLAM-1 similarto those found in the plasma of normal blood donors, a small butsignificant inhibition of leukocyte endothelial binding was observed.Circulating sLAM-1 is likely to be functional in vivo sinceimmunohistochemical staining of tissues with anti-LAM-1 mAb revealedsLAM-1 specifically bound to the luminal surface of endothelial cells,both on HEV-endothelium and at sites of inflammation.

Detection by Western Blot Analysis

In vitro, leukocytes and LAM-1 cDNA-transfected cells shed LAM-1 fromthe cell surface that can be detected in the culture supernatant fluid.To determine whether this process also occurs in vivo, the anti-LAM1-3mAb, which identifies an epitope within the lectin domain, was used toimmunoprecipitate reactive materials from normal human plasma. Theprecipitated materials were then analyzed by SDS-PAGE, transferred tonitrocellulose and the presence of a soluble form of LAM-1 wasvisualized by Western blot analysis using the anti-LAM1-14 mAb thatbinds to a LAM-1 epitope located within the SCR region. Two predominantisoforms of sLAM-1 were observed, a ˜62,000 M_(r) isoform and a75-100,000 M_(r) isoform. Analysis of the culture supernatant fluid fromPMA-stimulated lymphocytes, neutrophils and K562 cells transfected withLAM-1 cDNA (K562-LAM) revealed different, cell-specific isoforms ofsLAM-1. Neutrophils shed a 75-100,000 M_(r) isoform of sLAM-1, thattravelled as a broad band with an ill-defined upper border most likelydue to heavy glycosylation (Ord et al., J. Biol. Chem. 265:7760-7767(1990)). Lymphocytes generated a 62,000 M_(r) isoform and K562-LAM cellsshed a 71,000 M_(r) isoform of sLAM-1. No specific proteins wereisolated from the supernatant fluid of untransfected K562 cells usingthe above assays, and preclearing the plasma or supernatant fluid withthe anti-LAM1-3 mAb eliminated subsequent Western blot results.Therefore, it is likely that the two predominant isoforms of sLAM-1identified in plasma derived from both lymphocytes and neutrophils.Furthermore, sLAM-1 in serum contained the lectin, EGF and SCR domainsas it was visualized in these experiments using mAb reactive with thelectin (anti-LAM1-3) and SCR (anti-LAM1-14) domains, but the M_(r) ofsLAM-1 is smaller than that for LAM-1 isolated from detergentsolubilized cells as previously described (Lasky et al., Cell.56:1045-1055 (1989); Spertini et al., Leukemia 5:300-308 (1991)).

Quantitation of sLAM-1 in Plasma and Serum by ELISA

The amount of sLAM-1 found in human plasma was quantitated with asandwich ELISA using the anti-LAM1-5 mAb as a capture antibody andbiotinylated anti-LAM1-3 mAb as a detecting antibody. This specificcombination of mAb provided the highest level of sensitivity so that ˜5ng of sLAM-1 could be easily detected. The level of sLAM-1 determined byELISA in the sera of a population of healthy normal blood donors wasfound to be 1.6±0.81.0 μg/ml, n=63. In some donors, sLAM-1 levels weresimultaneously quantitated in plasma with similar results obtained(1.9±1.0 μg/ml, n=18). The possibility that sLAM-1 in plasma or serummight still be membrane bound was ruled out as the same levels of sLAMwere found in serum and plasma before and after ultracentrifugation.sLAM-1 was stable in whole blood left to stand at 20° C. beforeseparation of serum or plasma for up to at least 24 h. Storage of serumor plasma at 4° C. for up to three months in the presence of azide orrepeated thawing and freezing (up to ten times) did not affect theability to detect sLAM-1 in serum. It thus appears that sLAM-1 ispresent at relatively high levels in human plasma and serum.

Quantitation of sLAM-1 in Supernatants of Activated Leukocytes

Stimulation or the culturing of leukocytes has been associated withshedding of LAM-1 from the cell surface. In experiments carried out todetermine if sLAM-1 accumulated, culture supernatant fluid obtained froman erythroleukemia cell line transfected with the pLAM-1 cDNA (K562-LAM)was found to contain detectable sLAM-1, in contrast to supernatant fluidobtained from untransfected cells cultured at the same density.Similarly, medium from freshly isolated neutrophils cultured at 37° C.for 60 min also contained detectable sLAM-1. Incubation of theneutrophils with stimuli that do not affect cell surface LAM-1expression, granulocyte-colony stimulating factor, interleukin 1,monocyte-colony stimulating factor, interleukin 6, interferon γ, andinterleukin 4, did not induce an increase in sLAM-1. However,stimulation with formylated methionine-leucine-phenylalanine,lipopolysaccharide, granulocyte/monocyte-colony stimulating factor,interleukin 8, and TNF induced LAM-1 shedding corresponding to theirpotency to stimulate cell surface expression. Culturing lymphocytes for60 min at 37° C. caused some shedding of LAM-1, which was greatlyenhanced by PMA-treatment. Quantitation of the amount of sLAM-1 found inthe supernatant fluid following activation of neutrophils andlymphocytes (1×10⁷ cells/ml) varied between approximately 10 to 30ng/ml. However, PMA-activation of lymphocytes for greater than 25 min,and neutrophils for greater than 10 min, resulted in the gradualdegradation of sLAM-1.Degradation was not observed in the supernatantfrom K562-LAM cells, where sLAM was consistently found at concentrationsof 22±6 ng/ml (1.7±0.5% of standard plasma; n=14). Elevated levels ofsLAM-1 were also detected in the culture supernatant fluid oflymphocytes cultured with phytohemagglutinin, concanavalin A, andpokeweed mitogen for 3 to 6 d at 37° C. These mitogens are known tocause cell surface loss of LAM-1 (Tedder et al., J. Immunol. 144:532-540(1990)). Thus, loss of LAM-1 from the cell surface directly correlateswith an increase of sLAM-1 in the culture medium.

Inhibition of Lymphocyte Binding to Activated Endothelium by sLAM-1

sLAM-1 was semipurified from plasma by salt fractionation followed byaffinity chromatography with the anti-LAM1-3 mAb, as described. Thecolumn eluate was concentrated and the level of sLAM-1 present wasquantitated by ELISA and adjusted to ˜15 μg/ml (6 to 10% of total eluateprotein). sLAM-1, at different concentrations, in RPMI 1640/10% FCS wasincubated (15 min, 4° C.) with cytokine-activated endothelial cellsbefore examining lymphocyte attachment to endothelium through LAM-1.While sLAM-1 at physiological concentrations caused only partialinhibition of lymphocyte attachment (32.1±15.9%, n=10), it was found toinhibit most LAM-1-dependent lymphocyte binding at concentrations of 8μg/ml (52 to 100%) and caused almost total inhibition of LAM-1 mediatedbinding at 12 to 15 μg/ml (93 to 100%). Lymphocyte binding mediated byLAM-1 was calculated as the difference between total binding at anygiven concentration of sLAM-1 and lymphocyte binding found in thepresence of anti-LAM1-3 mAb which blocks all LAM-1 mediated adhesion(Spertini et al., J. Immunol. 147:2565-2573 (1991)). Importantly, sLAM-1at 15 μg/ml caused no additional inhibition of lymphocyte binding beyondthat obtained with anti-LAM1-3 mAb alone. When the sLAM-1 samples wereprecleared with the anti-LAM1-15 mAb (which does not block LAM-1function) before the assays, the preparation was not able to inhibitlymphocyte attachment demonstrating that sLAM-1 mediated the inhibition.Thus, it appears that sLAM-1 is capable of inhibiting LAM-1-specificlymphocyte adhesion to endothelium by binding to its putativeendothelial ligand.

The inhibitory capacity of sLAM-1 at normal physiological concentrations(1.5 μg/ml) was further evaluated to determine if it could alter theinteraction of lymphocytes with endothelium. Endothelium was activatedwith TNF (100 U/ml) for different periods of time to induce the LAM-1ligand in a manner similar to what might happen during the initiation ofan inflammatory response. During the course of induction of the LAM-1ligand, sLAM-1 treatment of the endothelium caused a consistent, butsmall, inhibition of LAM-1-dependent lymphocyte attachment (30% at 2 h,43% at 3 h, 41% at 4 h, 15% at 5 h and 22% at 6 h). Again, thecombination of sLAM-1 with the anti-LAM1-3 mAb did not cause a greaterinhibition than that observed with the mAb alone. Thus, during thedevelopment of an inflammatory response, it is likely that sLAM-1 willbe able to alter the course of leukocyte attachment, although theinfluence appears to be small in this in vitro assay.

sLAM-1 in Serum Blocks anti-LAM-1 mAb Binding

The presence of circulating sLAM-1 was further verified by demonstratingthat the reactivity of anti-LAM-1 mAb with LAM-1⁺ cells could beinhibited by plasma. When lymphocytes were stained using anti-LAM-1 mAbin undiluted human plasma, no significant staining was obtained atconcentrations of mAb that were saturating in RPMI/FCS. Next,lymphocytes (1×10⁶ /100 μl) were incubated with various concentrationsof LAM-1 directed mAb diluted (1:100) in autologous plasma or autologousplasma which had been precleared of sLAM-1 by immunoprecipitation. Aftercompletion of indirect immunofluorescence staining, antibody binding wasassessed by flow cytometry. In most cases, the presence of plasmainhibited anti-LAM-1 mAb staining when the mAb were used at 2 to 5μg/ml. Thus, ˜13 to 33 pM IgG₁ (MW 150,000) bound to ˜21 pM sLAM-1 (MW75,000) based on the concentration of sLAM present in plasma (1.6 μg/ml)determined as outlined above. sLAM-dependent inhibition of mAb bindingwas seen in plasma for mAb which recognize the lectin-domain(anti-LAM1-3, -4, and -10) and the EGF-like domain (anti-LAM1-5, and-15). In contrast, binding of the anti-LAM1-1 mAb, which binds anepitope in the EGF-like domain, was not significantly inhibited by thepresence of plasma.

The LAM1-1 Epitope Was Not Detected on sLAM-1 From Human Plasma

Since anti-LAM1-1 mAb binding to lymphocytes was not inhibited byplasma, the epitope identified by this mAb may not be present on sLAM-1.To examine this further, ELISA were performed in which differentLAM-1-directed mAb were bound to an ELISA plate as capture antibodieswith the anti-LAM1-3 mAb as the detecting antibody. Lectindomain-specific antibodies (anti-LAM1-6, -7, -10 and -11) and EGF-domainspecific mAb (anti-LAM1-5 and -15) gave strong, easily detectablepositive signals. Wells coated with the anti-LAM1-3 and -4 mAb, whichcross-block the binding of the detecting antibody (anti-LAM1-3), servedas internal controls for background reactivity. In contrast to all othermAb, anti-LAM1-1, gave a signal that was not significantly differentfrom background, as defined by using BSA as the capture reagent. Thus,the epitope located within the EGF-region which is specificallyrecognized by the anti-LAM1-1 mAb appears to be lost from LAM-1 aftershedding. As LAM-1 was easily recognized by all LAM-1-specific mAb inimmunofluorescence staining of lymphocytes and all three extracellulardomains are preserved on sLAM found in plasma, conformational changes inthe sLAM-1 protein may lead to the loss of the EGF-domain related LAM1-1epitope.

In order to determine whether the transmembrane or cytoplasmic regionsof LAM-1 are necessary to uphold the complete tertiary structure of itsextracellular domains, a second form of soluble LAM-1, a LAM-IgG fusionprotein was generated and epitope-mapped by ELISA. The chimeric cDNAused to generate the fusion protein was constructed so that it containedessentially the whole extracellular region of LAM-1. Culture supernatantfluid of COS cells that were transiently transfected with the chimericcDNA were tested for the production of LAM-IgG by ELISA. Theanti-LAM1-5, -7, and -15 mAb, which detected sLAM-1 in plasma and insupernatant fluid from PMA-stimulated K562-LAM cells, also bound theLAM-IgG chimera at similar levels. In contrast, while the anti-LAM1-1mAb failed to generate a significant signal with sLAM-1 from plasma andK562-LAM transfectants, it readily bound the LAM-IgG fusion protein.Thus, it is likely that sLAM-1 generated in vivo or in vitro loses anecessary conformational determinant that is required for anti-LAM1-1mAb binding.

Expression of LAM-1 in Human Inflammatory Tissues

Presumably, sLAM-1 is generated in vivo as leukocytes become activatedand/or transmigrate through endothelium. It has also been observed thatleukocytes isolated from various tissues express little LAM-1 (Tedder etal., J. Immunol. 144:532-540 (1990)), suggesting that as the cellstransmigrate the endothelium or localize in tissues, that they shedLAM-1. However, the presence of cell surface LAM-1 was not strictlyrestricted to leukocytes within blood vessels as revealed byimmunohistochemical analysis: Extravascular leukocytes positive forLAM-1 were seen in all tissues examined, and these included, wherepresent, lymphocytes, neutrophils and monocytes. The process ofextravasation does not result in complete loss of cell-surface LAM-1from all cells as lymphocytes which appeared to be traversing betweenendothelial cells remain LAM-1⁺, and some transmigrating monocytes alsoappeared LAM-1⁺. In general, monocyte/macrophages and granulocyteslocalized within tissues were largely LAM-1 negative, however, someextravasated neutrophils and macrophages were LAM-1⁺. Most importantly,focal staining of venular endothelial cells was observed with anti-LAM-1mAb in 2 of 4 hyperplastic lymph nodes, 2 of 3 sarcoid lymph nodes, on 1of 4 rheumatoid synovia, in 1 of 3 specimens of inflamed skin, and 2 of4 of appendicitis. This endothelial staining is consistent with thenotion that sLAM-1 is capable of binding to its ligand on the apicalsurface of endothelium.

Experimental Procedures Antibodies

LAM-1 directed mAb were the anti-LAM1-3, -4, -6, -7, -8, -10, -11, and-12 mAb directed against epitopes within the lectin domain, anti-LAM1-1,-5 and -15 reactive with epitopes within the EGF-like domain andanti-LAM1-14 which reacts with the SCR regions of LAM-1, all of the IgG₁isotype (Spertini et al., J. Immunol. 147:942-949 (1991)). Theanti-LAM-1 mAb were purified by salt fractionation followed by anionexchange chromatography, with the mAb concentration determined by lightabsorption. The anti-LAM1-3 mAb was bound to CNBr-activated Sepharose 4B(Pharmacia LKB Biotechnology, Piscataway, N.Y.) at 2.5 mg of mAb boundper ml of beads (anti-LAM-Sepharose) using the methods of themanufacturer.

Isolation of Blood Mononuclear Cells

Heparinized blood was obtained according to protocols approved by theHuman Protection Committee of Dana-Farber Cancer Institute, Boston,Mass. Mononuclear cells were isolated by Ficoll Hypaque density gradientcentrifugation. Cells were immediately suspended in RPMI 1640(Gibco-BRL, Gaithersburg, Md.) containing 101 FCS and kept at 4° C.until use. In most instances, these cells will be referred to aslymphocytes since 85 to 95% of the cell population was lymphocytes asdetermined by morphology (Wright's stain) and flow cytometry analysis.Neutrophils were purified by centrifugation on a cushion of Mono-PolyResolving Medium (Flow Laboratories, McLean, Va.) followed by lysis ofthe red cells with ice-cold hypotonic 0.2% (w/v) NaCl solution.Granulocytes were finally resuspended in HBSS containing 5% FCS (Sigma,St. Louis, Mo.). When sLAM was obtained for Western blot analysis, themononuclear cells were first incubated in plastic dishes for 30 min inRPMI/10% FCS at 37° C. to remove adherent monocytes. Nonadherent cellsretained surface LAM-1 and were predominantly lymphocytes (˜99%).

Cell Cultures

Lymphocytes were cultured in 24 well plates (Costar Corp., Cambridge,Mass.) at 10⁶ /ml in RPMI 1640 medium containing 10% FCS, 2%L-glutamine, penicillin, and streptomycin. The cells were cultured withPMA (100 ng/ml) for 60 min before the culture medium was harvested andtested for sLAM-1 by ELISA. Neutrophils were incubated in polypropylenetubes at 8×10⁶ cells/ml for 60 min at 37° C. either in HBSS/5% FCSmedium alone or containing granulocyte/monocyte-colony stimulatingfactor (25 ng/ml; a gift from Drs. Steven Clark and Gordon Wong,Genetics Institute, Cambridge, Mass.), monocyte-colony stimulatingfactor (100 ng/ml; Genetics Institute), tumor necrosis factor-a (TNF-α;100 U/ml; Genzyme Corp., Cambridge, Mass.), lipopolysaccharide (1 μg/ml;Escherichia coli 011:B4; Sigma), formyl-methionyl-leucyl-phenylalanine(10⁻⁸ M; Sigma), interferon-γ (1000 U/ml; Genzyme Corp., Cambridge,Mass.), or interleukin-1β (10 U/ml; Genzyme Corp., Cambridge, Mass.).After culture, the supernatants were tested for the presence of sLAM-1by ELISA. The human erythroleukemia cell line K562 was transfected withthe pLAM-1 cDNA as previously described (Tedder et al., J. Immunol.144:532-540 (1990)) and was be called K562-LAM throughout. These cellswere cultured in RPMI 1640/10% FCS and were kept at cell numbers between0.2 to 1×10⁶ cells/ml. All cells were incubated at 37° C. in 5% CO₂ with100% humidity.

Indirect Immunofluorescence Analysis

Indirect immunofluorescence analysis was carried out after washing thecells three times. Cells (1×10⁶ cells) were resuspended in 100 μl ofmedia containing various concentrations of the indicated mAb, andincubated for 60 min at 4° C. After washing, the cells were treated withFITC-conjugated goat anti-mouse Ig antibodies (Southern BiotechnologyAssociates, Birmingham, Ala.) for 20 min at 4° C. The cells were washed,and fixed (1% paraformaldehyde in PBS), and single color fluorescencewas determined on a flow cytometer (PROFILE™, Coulter Immunology,Hialeah, Fla.). Ten thousand cells were analyzed for each sample and themean peak channel number for positively stained cells was determinedusing a linear scale.

In some experiments, cells were stained in the presence of human plasma.sLAM was precleared from an aliquot of the same plasma byimmunoprecipitation with anti-LAM-Sepharose (1 ml of beads per 4 ml ofplasma). The efficiency of the immunoprecipitations was tested by ELISA(<20 ng/ml). Lymphocytes (1×10⁶) were resuspended either in 100 μl ofplasma or precleared plasma containing various concentrations of thepurified mAb (added 1:100) as indicated. The cells were washed twice,stained and analyzed as above.

Endothelial-leukocyte Attachment Assay

Lymphocyte adhesion to cytokine-activated endothelium under non-staticconditions was determined in a test system adapted from theStamper/Woodruff assay for frozen tissue sections (Stamper Jr. et al.,J. Exp. Med. 144:828-833 (1976)) exactly as described (Spertini et al.,J. Immunol. 147:2565-2573 (1991)). Briefly, human umbilical veinendothelial cells (HUVEC) were isolated from cord veins, and grown inM199 medium supplemented with 10% FCS, endothelial cell growth factor(50 μg/ml, Biomedical Technologies, Inc., Stoughton, Mass.) and porcineintestinal heparin (50 mg/ml; Sigma) as described (Spertini et al., J.Immunol. 147:2565-2573 (1991)). Endothelial cells were grown toconfluence on gelatin (0.1%) coated glass slides and stimulated withTNF-α (100 U/ml) at 37° C. for the times indicated. The monolayers werecarefully washed, and incubated at 4° C. for 15 min with 75 μl ofRPMI/10% FCS alone or containing semipurified sLAM-1. As a control, insome instances media containing semipurified sLAM-1 were precleared byimmunoprecipitation with anti-LAM-Sepharose. Without further washing,5×10⁶ lymphocytes in 75 μl of the respective media were added. After 20minutes of incubation at 4° C. with rotation at 64 rpm, the slides wererinsed, then fixed overnight in glutaraldehyde, (1% (v/v) in PBS;Polysciences, Warrington, Pa.), and stained with hematoxylin. The numberof adherent leukocytes was determined by counting 6 microscopic fields(0.09 mm² /field) and the results were expressed as means ±SD.

Immunohistochemistry

Blocks of fresh human tissue were snap frozen. The specimens wereperipheral lymph node (non-specific hyperplasia, 4 cases), sarcoid lymphnode (3 cases), acute appendicitis (4 cases), rheumatoid synovium (4cases), and inflamed skin (insect bite, delayed hypersensitivityreaction to tuberculin, and pityriasis lichenoides chronica; 1 caseeach). Immunohistochemistry was performed on cryostat sections using theanti-LAM1-3 mAb and an avidin-biotin-peroxidase method withdiaminobenzidine (Rice et al., Am. J. Pathol. 138:385-393 (1991)). As acontrol, a murine IgG₁ (Coulter Immunology, Hialeah, Fla.) of irrelevantspecificity was used at a concentration three times stronger than thetest mAb. The control IgG did not produce detectable staining and thusall stated results refer to specific staining only obtained with thetest mAb.

Production of the LAM-1 and IgG Chimera cDNA and Protein

The 1400-bp BanII fragment from a cDNA encoding the CH1 through CH3domains of the human IgG₁ constant region was inserted at a BanII siteintroduced into pLAM-1 cDNA (Chin et al., J. Immunol. 125:1770-1774(1980)) by oligonucleotide directed mutagenesis. The exchange ofnucleotides in pLAM-1 cDNA from GAGGGT¹⁰⁷⁸ to GAGCCC created a BanIIsite corresponding to amino acid number 370 in the membrane proximalregion of the mature protein. The antisense oligonucleotide used togenerate the new restriction site, plus a sense oligonucleotide anchorfrom the plasmid 5' end of the pLAM-1 cDNA were used to amplify the 5'end of pLAM-1 cDNA. The PCR product was treated with T4 kinase, gelpurified, subcloned into pSP65 (Promega Biotech, Madison, Wis.) anddigested with BanII and Kpnl. In parallel, the LAM-1 cDNA subcloned intopSP65 was digested with Kpnl and Pvul, and a cDNA encoding human heavychain IgG₁ was digested with BanII and Pvul. After gel purification, theDNA fragments thus obtained were mixed with the PCR product previouslydigested with Kpnl and BanII and ligated together. The PCR product wassequenced, and the conservation of LAM-1 and IgG₁ restriction sites inthe pLAM-IgG₁ DNA was confirmed by restriction mapping. The LAM-IgG₁ DNAwas subcloned into the Ap^(r) M8 expression vector (provided by Dr.Lloyd Klickstein, Center for Blood Research, Boston, Mass.) and used totransiently transfect COS cells by the DEAE dextran method. Thetransfected COS cells were cultured in AIM-V serum-free media(Gibco-BRL0 and the supernatant fluid containing the chimeric LAM-1/IgG₁fusion protein was harvested after 3 d.

Soluble LAM-1 Purification

sLAM-1 was semipurified from plasma obtained from heparinized humanblood. Plasma was salt-fractionated with Na₂ SO₄ (18% w/v) before thesLAM-1 containing supernatant fraction was dialyzed against 0.02M Trisbuffer (pH 8.0), 0.5M NaCl. The sLAM-1 preparation was further purifiedby affinity column chromatography using anti-LAM-Sepharose. sLAM-1 waseluted from the column with 0.1M Na acetate buffer (pH 3.5), 0.15M NaCland the low pH of the eluate was immediately raised by the addition of2.0M Tris buffer (pH 9.0). The pooled fractions of the eluate peak wereultrafiltrated and resuspended in PBS. The concentration of sLAM-1 wasdetermined by ELISA and subsequently adjusted to approximately 15 μg/mlin PBS. At this sLAM-1 concentration, the total protein concentration ofthe samples varied between 130 to 220 μg/ml as determined by lightabsorption, and sLAM-1 represented ˜6 to 10% of total protein. Ingeneral, this procedure gave a 2200 to 3700 fold enrichment for sLAM-1.For use in lymphocyte-endothelial adhesion assays, semipurified sLAM-1was transferred into RPMI 1640/10% FCS by further ultrafiltration.

Western Blot Analysis

sLAM-1 was semipurified from plasma as described above and furtherpurified by immunoprecipitation using anti-LAM-Sepharose with repeatedwashing of the beads in alternating high salt (0.5M NaCl, 0.2%Na-deoxycholate) and low salt (0.125M NaCl, 0.05% Na-deoxycholate) RIPAbuffer (100 mM Tris pH 8.0, 1% (v/v) Triton X-100, 10 mM EDTA, 10 mMEGTA, 10 mM NaF, 1 mg/ml BSA). Proteins were eluted from the beads with0.1M acetate buffer (pH 3.5), 0.15M NaCl. Supernatant fluid fromPMA-stimulated cells was also analyzed. Cells (1×10⁷ /ml) including,neutrophils (10 ng/ml PMA in RPMI 1640 for 10 min at 37° C.),lymphocytes (10 ng/ml PMA in RPMI 1640 for 25 min at 37° C.) andK562-LAM transfectants (100 ng/ml PMA in PBS for 120 min at 37° C.) wereinduced to shed essentially all detectable cell surface LAM-1, beforebeing pelleted by centrifugation (4° C., 400×g, 10 min). The supernatantfluid was saved and concentrated ten fold by ultrafiltration (AmiconCorp., Danvers, Mass.). Protein samples (100 μl) were applied to a 7.5%SDS-polyacrylamide gel, electrophoresed and blotted onto nitrocellulose.Western blot analysis was performed using the anti-LAM1-14 mAb (ascites,1:2000) as the antigen detecting antibody. The blot was developed usingalkaline phosphatase conjugated goat-anti-mouse IgG₁ antibody (SouthernBiotechnology Associates) and NBT/BCIP as substrate (Promega, Madison,Wis.). In preliminary experiments, the anti-LAM1-14 mAb was the mostsensitive of the twelve anti-LAM-1 mAb tested, of which anti-LAM1-3, -4,-8, -10, -14 and -15mAb were found to give positive staining.

Soluble LAM-1 ELISA

Wells of microtiter plates (96 well, flat bottom, E.I.A./R.I.A. plate,Costar, Cambridge, Mass.) were coated with anti-LAM-1 mAb (100 μg/ml) in0.1M borate buffer, pH 8.4 at 4° C. for 18 h. Following two washes withTris buffered saline (TBS; 20 mM Tris pH 7.5, 0.5M NaCl) the wells wereblocked with 100 μl of 2% bovine serum albumin and 1% gelatin in TBS for1 h at 37° C. The wells were washed three times with TBS containing0.05% Tween 20 (TBST), and the test samples diluted into TBST (50 μl)were added to triplicate wells for 90 min at 20° C. Each assay includedthe titration of a previously quantified standard plasma sample that wasused to generate a standard dilution curve. Dilution into fetal calfserum or pig serum had no significant effect on results. After beingwashed four times with TBST, the plates were incubated with 100 μl ofbiotinylated anti-LAM1-3 mAb (1 μg/ml) in TBST for 60 min at 20° C.After another four washes, 100 μl of avidin-horseradish peroxidase (0.1μg/ml, Pierce, Rockford, Ill.) was added for 30 min at 20° C. After fourmore washes with TBST, the plates were finally developed using 100 μl ofo-phenylenediamine (0.125% w/v, Sigma Chemical Co.) as a substrate in0.1M citrate buffer, pH 4.5 in the presence of 0.015% H₂ O₂. The OD ofthe reaction mixture was quantitated using an ELISA-reader (v-max,kinetic microplate reader, Molecular Devices, Menlo Park, Calif.).Results were obtained when the OD for the well containing the highestconcentration of standard plasma was ˜0.8 at 495 nM. Background ODvalues were obtained using wells coated with albumin only. The ELISA wasmade quantitative by using a standard plasma to generate a titrationcurve for each assay. The relative concentration of sLAM-1 in individualsamples was calculated by comparing the mean OD obtained for triplicatewells to a semilog standard curve of titrated standard plasma usinglinear regression analysis. Sample concentrations were obtained byinterpolation of their absorbance on the standard curve.

The amount of sLAM-1 present in the standard plasma was quantitated intwo ways. First, K562-LAM-1 cells (˜11 L of cultured cells, ˜1.1×10¹⁰cells) were resuspended in PBS (1×10⁷ cells/ml) and were stimulated withPMA (100 ng/ml) for 2 h at 37° C. The supernatant fluid was collected,concentrated by ultrafiltration and affinity purified by columnchromatography using anti-LAM-Sepharose. The semipurified sample waselectrophoresed on a 10% SDS-polyacrylamide gel that was subsequentlystained by Coomassie-blue to reveal a prominent band of 71,000 M_(r) andadditional bands of ˜180-, 57-, 47-, and 22,000 M_(r). The destained gelwas scanned using a Hewlett Packard Desk Scanner and the density of the71,000 M_(r) band was quantitated against a standard curve generatedwith BSA using the Enhance™ program (Microsystems, Des Moines, Iowa) onan Apple Macintosh IIcx computer. The concentration of sLAM-1 in thestandard plasma was calculated to be ˜1.3 μg/ml by comparing the signalfrom semipurified sLAM-1 to the LAM-ELISA titration curve of standardplasma with linear regression analysis. The detection limit of theLAM-ELISA was determined to be ≧5 ng/ml. In a second set of experiments,COS cells were grown in serum free medium after transient transfectionwith the LAM-IgG chimera cDNA. Supernatant fluid was collected from thecells and run over a Protein-A Sepharose (Pharmacia LKB Biotechnology)affinity chromatography column, and the fusion protein was eluted fromthe column by high salt-low pH buffer. The purified fusion protein wasquantitated after SDS-PAGE analysis by comparison of the stained proteinband with a standard curve of BSA. From this analysis, it appeared thatOD values for standard plasma would be equivalent to ˜1.9 μg/ml ofLAM-IgG fusion protein. Since the dimeric nature of the fusion proteinmight double the intensity of staining in our sandwich ELISA, the amountof sLAM-1 in the standard plasma may be half the value of ˜1.9 μg/ml.

Test samples of human plasma, pig serum, and culture supernatant fluidfrom K562-LAM cells, LAM-IgG cDNA-transfected COS cells and mocktransfected COS cells were examined in the ELISA for the presence ofsLAM-1. Significant reactivity was only observed in the human plasma,and supernatant fluid from K562-LAM cells and LAM-IgG cDNA transfectedcells. In ten assays, the LAM-ELISA had a linear correlation coefficient≧0.97 using a titration curve of standard plasma over a range of 5-1300ng/ml. The interassay coefficient of variation for measuring sLAM-1levels in plasma on three different days was 4.5%. There was nosignificant difference in sLAM-1 levels found between samples afterfreezing or after freezing-thawing up to ten times and no apparentdecrease in ability to quantitate sLAM-1 amounts in whole blood left atroom temperature for several hours. Therefore, sLAM-1 was quite stablein whole blood.

The amount of sLAM-1 found in a population of normal individuals wasdetermined for serum (1.92±0.96 μg/ml, n=18). Plasma was alsosimultaneously isolated from the same individuals and quantitated in theLAM-ELISA, which gave average sLAM-1 levels of 1.91±0.98 μg/ml. sLAM-1could also be detected in tissue culture supernatant fluid. Followinglymphocyte activation with mitogens, increased levels of supernatantfluid sLAM-1 were detected by day 1 and levels increased over a 6-dayculture period.

Use

Monoclonal antibodies that do not identify shed receptor LAM-1 yetidentify the cell-surface receptor can be used as therapeutics fordirect administration to patients. The use of antibodies of theinvention will prevent many of the secondary side effects of antibodyadministration that may result from immune complex formation andcross-linking or binding of antibody to the soluble form of thereceptor. Also, since the shed form of the receptor is a naturallyoccurring molecule present in high levels, it is possible to designrecombinant protein products that are similar in structure to sLAM-1;i.e., a recombinant protein without a membrane spanning region and acytoplasmic tail would mimic the natural serum protein. Thischaracteristic would diminish the immunogenicity of such a recombinantto the species of origin and allow use of the receptor as a therapeuticproduct as well. Minor changes to the primary amino acid sequence of thereceptor in the ligand binding region could be introduced to induce ahigher binding constant or affinity for ligand. This modified, truncatedrecombinant protein could then be used in soluble form as a therapeuticwith low immunogenicity yet a higher capacity to bind and block ligandbinding of the cell-surface receptor protein.

Antibodies of the invention will also be useful as diagnostic agents.For example, the ability to identify leukocytes that expresscell-surface LAM-1 is important for determining the migration potentialof a given population of cells and the state of cellular differentiationand activation. Because of the high levels of sLAM-1, it will bedifficult to use antibodies in the presence of biological fluids toidentify the cell-surface molecule. However, antibodies like anti-LAM1-1will be useful for identifying receptors present only on thecell-surface since they do not react with shed receptor in blood orbiological fluid samples. For example, whole blood indirectimmunofluorescence analysis will be facilitated and the in vivo imagingor analysis of receptor distribution will be possible with significantlylower amounts of antibody.

Also, monitoring of the levels of sLAM-1 could provide diagnostic orprognostic information relating to inflammatory disorders, leukocytemobilization, malignancy or infection. For example, since levels of sLAMwere easily and accurately quantitated in serum from normal individuals,serum levels of sLAM-1 were measured in patients with various states ofsystemic inflammation or infection. While patients with severe burns hadsLAM-1 levels similar to those found in the normal population, serumfrom Kawasaki syndrome patients had sLAM levels generally less thanthose of normals. In contrast, patients suffering from sepsis orHIV-infection showed markedly elevated levels of sLAM-1 in serum thatwas significantly different (p<0.005) from that of a population ofnormal blood donors. sLAM-1 was also detected in cerebrospinal fluid,but the levels of sLAM-1 were generally only 0.6 to 6% of those found innormal serum. Thus, altered sLAM-1 levels, as an indication of leukocytemobilization or activation, have diagnostic or prognostic value forinflammatory or infectious disorders.

The assays described herein demonstrate that detection of sLAM-1 levelsin solution is readily achievable. Furthermore, quantitation of sLAM-1levels in a very precise manner is also achievable, even when the sLAM-1is contained within a biological fluid. Also revealed is thatquantitation of sLAM-1 levels in biological fluids from patients mayhave diagnostic value.

One of ordinary skill, after reading the foregoing descriptions will beable to effect a variety of means for detecting not only sLAM-1, butalso the parent protein LAM-1 or any fragment of LAM-1. Detaileddescriptions of two binding assays have been provided, Western blotanalysis and an antibody-based ELISA. However, other agents which bindselectively to LAM-1 may be used to capture or reveal the presence ofLAM-1, or fragments thereof, such as carbohydrate moieties, includingPPME, fucoidin or other LAM-1 ligands. Cell based capture mechanismssuch as cytokine-activated endothelium or tissue sections that containthe LAM-1 ligand could also serve as binding agents.

In addition, the capture of LAM-1 or fragments thereof can be assessedby a variety of means obvious to those with skill in the art. Forexample, alterations in the structure, resonance or optical propertiesof the capture reagent or detecting reagent can be measured to revealLAM-1 binding. The capture reagent may also be a polyclonal antiserumspecific for LAM-1 or fragments thereof. Detection reagents can also belabeled using radioactive, immunoreactive, or enzymatically activeagents or compounds to visualize binding. In addition, the capturereagent may not be specific for LAM-1 or fragments thereof, but LAM-1specificity may be obtained through the use of LAM-1 specific detectingreagents, or the detecting reagent may be non-specific and the capturereagent specific.

Quantitation of the amount of LAM-1, or fragments thereof, present in asample can also be achieved using a variety of means. For example,recombinant LAM-1 protein or fragments can be used as a standard as wasdemonstrated in the assay for naturally occurring sLAM-1 in serum orplasma. Also, LAM-1 or fragments thereof produced in tissue culturesupernatant fluid of mammalian, bacterial or insect cells can be used.Even small peptide fragments which will bind to the capture reagent canbe used to assess and quantitate LAM-1 binding in a competitive assay,or the displacement of small molecules from the capture reagent by LAM-1can be used to quantitate the amount of LAM-1 present.

While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

Deposits

Hybridoma LAM1-1 was deposited on Jul. 28, 1991, with the American TypeCulture Collection (ATCC) as ATCC No. HB10844.

Applicants' assignee, Dana-Farber Cancer Institute, Inc., representsthat the ATCC is a depository affording permanence of the deposit andready accessibility thereto by the public if a patent is granted. Allrestrictions on the availability to the public of the material sodeposited will be irrevocably removed upon the granting of a patent. Thematerial will be available during the pendency of the patent applicationto one determined by the Commissioner to be entitled thereto under 37CFR 1.14 and 35 USC 122. The deposited material will be maintained withall the care necessary to keep it viable and uncontaminated for a periodof at least five years after the most recent request for the furnishingof a sample of the deposited microorganism, and in any case, for aperiod of at least thirty (30) years after the date of deposit or forthe enforceable life of the patent, whichever period is longer.Applicants' assignee acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

What is claimed is:
 1. A method for blocking LAM-1 dependent cellular adhesion in the presence of shed LAM-1, comprising the step of contacting a population of cells expressing said LAM-1 with an effective amount of a monoclonal antibody that is reactive with cell-surface bound LAM-1 but not reactive with shed LAM-1.
 2. A method for inhibiting leukocyte migration into tissues, comprising the step of administering an amount of a monoclonal antibody that is reactive with cell-surface bound LAM-1 but not reactive with shed LAM-1, effective to block LAM-1 dependent cellular adhesion in the presence of shed LAM-1. 